RCA-free Adenovirus Amplification Service using 293RCA- Packaging Cell Line
Excellgen's 293RCA- cell line is an E1-complementing cell line that eliminates the occurrence of Replication Competent Adenoviruses (RCA). First generation E1-deleted recombinant adenoviruses are typically propagated in complementing Human Embryonic Kidney HEK-293 cell line which stably expresses the E1 gene products (Graham et al., 1977). RCAs are known to occur after several passages of amplification in HEK-293 cells. RCAs emerge following a double crossover event between the homologous overlapping sequences in the rAd genome and the E1 genes from the 293 cells, resulting in the loss of transgene and its replacement by E1 (Lochmüller et al., 1994, Hehir et al., 1996, Zhu et al., 1999). The presence of RCAs in adenovirus stocks could have deleterious effects to the cells when used in gene delivery experiments in vitro, could be harmful to animals used for in vivo studies and pose potential risks to patients by activating the immune system or by acting as helper viruses for the replication of attenuated viral vectors (Imler et al., 1995).
The 293RCA- cell line was engineered by Excellgen and Feldan to express the E1 genes without overlapping sequences with the adenovirus backbone (such as AdEasy™), thus eliminating the risk of generating RCAs in viral stocks. The new 293RCA- cell line provides scientists involved in functional genomics studies, gene delivery and gene therapy research with better control over RCAs in their viral stocks.
Features
· No homologous sequences with adenoviral backbones
· No risk of occurrence of RCA contaminants
· Affordable alternative for RCA-free cultures
Services available with the 293RCA- cell line
· Recombinant Adenovirus construction
· Plaque purification
· Amplification
· Purification
· Titration
· RCA detection: PCR- and cell-based
References
Hehir et al. 1996. Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence. J. Virol. 70: 8459-8467.
Imler et al. 1995. Trans-complementation of E1-deleted adenovirus: A new vector to reduce the possibility of codissemination of wild-type and recombinant adenoviruses. Hum. Gen. Ther. 6: 711-721.
Lochmller et al. 1994. Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (dE1 dE3) during multiple passages in 293A cells. Hum. Gene Ther. 5: 1485-1491.
Zhu et al. 1999. Characterization of replication-competent adenovirus isolates from large-scale production of a recombinant adenoviral vector. Hum. Gene Ther. 10:113-121.